Hepatic stellate cells (HSCs), previously known as Ito cells, lipocytes, perisinusoidal cells or fat-storing cells, are a minor cell type (roughly 5-8% of total liver cells) most commonly found in the sinusoidal area of adult livers. The basic pathobiology and history of HSC discovery have been reviewed elsewhere (Burt, (1999), J. Gastroenterol. 34(3):299; Sato et al. (2003), Cell Struct Funct. 28(2):105). The major physiological functions of HSC include fat storage, vitamin A uptake and metabolism, and the production of extracellular matrix proteins. In the past decade, HSCs have been implicated in mounting a defense during hepatic injury, and mediating hepatic fibrogenesis by over-producing pro-fibrotic cytokines and consequently extracellular matrix (ECM) molecules. HSCs are believed to play a role in the pathogenesis of a number of clinically important conditions such as, for example, hepatic fibrosis, cirrhosis, portal hypertension and liver cancer (Geerts (2004), J. Hepatol. 40(2):331). Hence, HSCs have also become a target for the development of anti-fibrotic therapies (Bataller et al., (2001), Semin Liver Dis. 21(3):437; Bataller et al., (2005), J. Clin Invest. 115(2):209; Friedman (2003), J. Hepatol. 38 Suppl 1:S38).
Activation of HSCs is a dominant event in fibrogenesis. During activation, quiescent vitamin A storing cells are converted into proliferative, fibrogenic, proinflammatory and contractile ‘myofibroblasts’ (Friedman (2003), J. Hepatol. 38 Suppl 1:S38; Bataller et al. (2005), J. Clin Invest. 115(2):209; Cassiman et al. (2002), J. Hepatol. 36(2):200). HSC activation proceeds along a continuum that involves progressive changes in cellular function. In vivo, activated HSCs migrate and accumulate at the sites of tissue repair, secreting large amounts of ECM components and regulating ECM degradation (Cassiman et al. (2002), J. Hepatol. 36(2):200). HSC identity both in vitro and in vivo has been traditionally identified with antibodies. Initially, a polyclonal rabbit antibody against chicken desmin (an intermediate filament) was used to stain cells with stellate shape in liver slices and skeletal myofibrils in rat (Yokoi et al. (1984), Hepatology. 4:709). Additional antibodies against vimentin (another intermediate filament) and smooth muscle-alpha-actin (SMAA) were subsequently employed to study liver fibrosis in rat (Bhunchet et al. (1992), Hepatology. 16:1452; Baroni et al. (1996), Hepatology. 23(5):1189). Despite their poor tissue- and cell-specificity, these three markers (desmin, vimentin and SMAA) have remained a common battery for identifying HSCs. Glial fibrillary acidic protein (GFAP) has also been indicated to be a marker for HSCs (Buniatian et al. (1996), Eur J Cell Biol. 70(1):23; Levy et al. (1999), Hepatology 29(6):1768; Cassiman et al., (2002), J. Hepatol. 36(2):200; Xu et al. (2005), Gut. 54:142).
Cell specific promoters are of great interest to those involved in genetic engineering for their potential to drive expression of a target gene in a specific subpopulation or subset of cells either in vitro or in vivo.
Several promoters have been investigated for their ability to express a gene of interest specifically in HSCs in vitro and in vivo. These promoters include the human collagen alpha 1 (ColI; Slack et al. (1991), Mol Cell Biol. 11(4):2066; Brenner et al. (1993), Hepatology 7(2):287; Yata et al. (2003), Hepatology 37:267), SMAA (Magness et al. (2004), Hepatology 40:1151), LIM domain protein CRP2 (CSRP2), tissue inhibitor of metalloproteinase-1 (TIMP-1) and smooth muscle-specific 22-kDa protein (SM22alpha) (Bahr et al. (1999), Hepatology 29(3):839; Herrmann et al. (2004), Liver International 24: 69).
In astrocytes, a fragment of the human GFAP (hGFAP) promoter has been shown to drive expression of operatively coupled transgenes in vitro and in vivo. The activity of this promoter fragment in non-astrocytic cells has been shown to be less predictable. The promoter fragment unreliably expressed lacZ in Müller cells in transgenic mice lines, leading to the suggestion that Müller cells may require regulatory elements beyond those contained in the promoter fragment (Brenner (1994), J Neurosci. 14: 1030). In Schwann cells, the transcription initiation site of the endogenous GFAP promoter is 169 nucleotides upstream from the transcription initiator site in astrocytes (Feinsten et al. (1992) J. Neurosci Res. 32(1):1). Further, while Schwann cells are known to express endogenous GFAP, these cells are also unreliably labeled in hGFAP-LacZ transgenic mice (Zhuo (1997), Developmental Biology 187:36).